EP2527351B1 - Lipophosphonoxins, method of their preparation and use - Google Patents
Lipophosphonoxins, method of their preparation and use Download PDFInfo
- Publication number
- EP2527351B1 EP2527351B1 EP12169491.3A EP12169491A EP2527351B1 EP 2527351 B1 EP2527351 B1 EP 2527351B1 EP 12169491 A EP12169491 A EP 12169491A EP 2527351 B1 EP2527351 B1 EP 2527351B1
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- EP
- European Patent Office
- Prior art keywords
- diastereomers
- mmol
- group
- general formula
- mixture
- Prior art date
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- 238000000034 method Methods 0.000 title claims description 18
- 238000002360 preparation method Methods 0.000 title claims description 10
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- ULXTYUPMJXVUHQ-OVTFQNCVSA-N lipid II Chemical compound OC(=O)[C@@H](C)NC(=O)[C@@H](C)NC(=O)[C@H](CCCCN)NC(=O)CC[C@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@@H]1[C@@H](NC(C)=O)[C@@H](OP(O)(=O)OP(O)(=O)OC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 ULXTYUPMJXVUHQ-OVTFQNCVSA-N 0.000 description 1
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- JPFWJDMDPLEUBD-ITJAGOAWSA-N polyoxorim Polymers O[C@@H]1[C@H](O)[C@@H]([C@H](NC(=O)[C@H]([C@H](O)[C@@H](O)COC(N)=O)N)C(O)=O)O[C@H]1N1C(=O)NC(=O)C(C(O)=O)=C1 JPFWJDMDPLEUBD-ITJAGOAWSA-N 0.000 description 1
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
Definitions
- the invention relates to novel compounds with antibacterial properties, methods of synthesis thereof, and utilisation thereof in vitro and in vivo.
- phosphonoxin (1) An effective inhibitor of Giardia trophozoite growth termed phosphonoxin (1) ( Figure 1 ) with an activity that rivaled existing therapeutics was recently reported.
- Phosphonoxin was designed as a transient-state inhibitor of a glycosyl transferase - cyst wall synthase (CWS).
- CWS catalyzes synthesis of the chitin-like poly ⁇ -1-3-linked N -acetylgalactosamine [poly(GalNAc)] that comprises about 63% of the giardia cyst wall.
- poly(GalNAc) glycosyl transferase - cyst wall synthase
- phosphonoxin did not specifically inhibit cyst formation, it potently inhibited vegetative growth.
- the molecule of phosphonoxin display certain structural similarity to several types of nucleoside antibiotics: (i) polyoxins 3, (ii) muraymycins 4, and (iii) caprazamycins 5.
- the polyoxins are a new group of antifungal antibiotics isolated from Streptomyces cacaoi that inhibit the growth of a number of mycelial fungi by interfering with chitin synthesis.
- Polyoxin D shares a gross structural similarity with uridine diphospho- N -acetyl-D-glucosamine (UDP-GlcNAc 2) in agreement with its role as a competitive inhibitor.
- the muraymycins isolated from a culture broth of Streptomyces species are members of a class of naturally occurring 6'- N -alkyl-5'- ⁇ - O -aminorybosyl- C -glycyluridine antibiotics.
- the muraymycins bearing lipophylic side chain exhibit excellent activity against gram-positive bacteria.
- the muraymycins inhibit the formation of lipid II and peptidoglycan and are believed to be inhibitors of phosphor-MurNAc-pentapeptide translocase (MraY), which is responsible for the formation of lipid I in the peptidoglycan biosynthesis pathway.
- the caprazamycins (CPZs) ( 5 ) were isolated from the culture broth of the actinomycete strain Streptomyces spp. MK730-62F2 in 2003 ( Igarashi M. et al., J. Antibiot. 2003, 56, 580 ; Igarashi M. et al., J. Antibiot. 2005, 58, 327 ) and represent the most recent members of the class of naturally occurring 6'- N -alkyl-5'- ⁇ - O -aminoribosyl- C -glycyluridine antibiotics that include the liposidomycins (7) (LPMs).
- LPMs liposidomycins
- This invention provides novel compound of general formula I that exhibit significant antibacterial activity, in particular against gram-positive bacteria.
- the advantages of these compounds include simple synthesis and modular structure allowing for further optimization of their biological properties.
- An object of this invention are lipophosphonoxins of general formula I
- Compounds of formula I contain several chiral centers (particularly on the phosphorus atom and in the R 3 group). The presence of a chiral center allows a compound to exist as one of two possible optical isomers ((R)- or (S)-enantiomer), or as a racemic mixture of both. In this case, where several chiral centers are present, diastereoisomers and mixtures of diastereoisomers are obtained, which are all covered by general formula I and included in the scope of this invention.
- Another object of this invention is the synthesis of the novel lipophosphonoxins.
- the starting material for the synthesis of the final compounds arc nucleoside vinylphosphonic acids of general formula 11 (Scheme 2). These compounds are prepared by esterification of methyl vinylphosphonate 9 ( Gao, Feng et al.; Chemistry-A European Journal, 2009, 15, 9, 2064 ⁇ 2070 ) that is prepared according to literature procedures.
- the synthesis according to this invention is novel, simpler than the previously described procedures and affords good yields.
- the commercially available dimethyl vinylphosphonate VIII (see Scheme 1) is heated overnight in 40% - 70% aqueous pyridine at 50 - 80 °C.
- methyl vinylphosphonate IX is allowed to react in the second reaction step B with nucleoside X possessing free 5'-hydroxyl group and protected in the positions 2'and 3'with a group selected from 2',3'-isopropylidene, 2',3'-bis(dimethoxytrityl), 2',3'-dibenzoyl, and 2',3'-diacetyl.
- the esterification is carried out using triisopropylbenzenesulfonylchloride (TPSCl) under N- methylimidazole catalysis in a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran (THF), dichloroethane, chloroform and dichloromethane.
- TPSCl triisopropylbenzenesulfonylchloride
- a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran (THF), dichloroethane, chloroform and dichloromethane a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran (THF), dichloroethane, chloroform and dichloromethane.
- the obtained methyl ester is purified by chromatography on silica gel using linear gradient of ethanol in chloroform.
- the vinylphosphonic acids XI are esterified with alcohol using TPSCl as a condensing agent under N -methylimidazole catalysis in a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran (THF), dichloroethane, chloroform and dichloromethane (Scheme 3).
- TPSCl tetrahydrofuran
- the obtained vinylphosphonate XII is purified by chromatography on silica gel using linear gradient of ethanol in chloroform.
- the final step D) consists in Michael addition of secondary amine to the vinyl moiety of intermediate XII.
- the reaction is accomplished by heating in a solvent selected from the group comprising acetonitrile, dioxane, THF, dichloroethane, chloroform, ethanol, propanol, isopropanol and n-butanol at 80 - 120 °C for 4 to 12 hours.
- the protecting groups are removed from the nucleoside moiety.
- the final product is isolated by chromatography on silica gel using linear gradient of mixture H1 (as described above) in ethyl acetate. If required, the final product is re-purified by preparative HPLC on reverse phase.
- the Michael addition in the step D is carried out in n-butanol at 105 °C; more preferably, the reaction is perfomed overnight.
- the esterification in the steps B and/or C is carried out preferably in dichloromethane.
- the nucleoside protecting groups are removed by ethanolic methylamine or aqueous ammonia: if 2',3'-protected ribonucleoside is used, the protecting group is preferably removed by treatment with 0.5 mol.l -1 hydrochloride in methanol overnight at room temperature.
- Another object of the present invention are the lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds for use as medicaments.
- Yet another object of the present invention are the lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds for use as antibacterial medicaments.
- Another object of the present invention is an antibacterial medicament containing as the active ingredient at least one lipophoshonoxin of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixture of such compounds.
- an object of the invention is the use of lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds as active components of disinfectants and/or selective growth media for in vitro cultivations.
- the compounds according to this invention possess antibacterial activity particularly against bacterial strains Enterococcus faecalis, Bacterium subtilis, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus haemolyticus, Enterococcus faecium, Staphylococcus epidermidis, including strains resistant to current antibiotics.
- the compounds according to this invention simultaneously exhibited none or very low effect on the viability of normal human erythroid progenitor cells cultivated in vitro in the range of concentrations sufficient for antibacterial activity. The same applies to cytotoxicity induced by these compounds.
- the title product is obtained by flash chromatography on silica gel using linear gradient of solvent mixture H1 (ethyl acetate:acetone:ethanol:water, volume ratios 4:1:1:1) in ethyl acetate in 48% yield (11.38 g, 23.93 mmol ) as a yellowish foam.
- TPSCl (4.68 g, 15.45 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.93 g, 5.15 mmol), tetradekanol (2.2 g, 10.31 mmol), and N-methylimidazole (1.22 ml, 15.45 mmol) in DCM (50 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- TPSCI (4.12 g, 13.62 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.7 g, 4.54 mmol), hexadecanol (2.9 g, 12 mmol), and 1-methylimidazole (1.08 ml, 13.62 mmol) in DCM (50 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- the organic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 50% yield (1.38 g, 2.3 mmol) in the form of colorless viscous oil.
- TPSCl (4.16 g, 13.74 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.7 g, 4.54 mmol), octadecanol (2.48 g, 9.17 mmol), and 1-methylimidazole (1.08 ml, 13.74 mmol) in DCM (50 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- the rganic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 43% yield (1,23 g, 1,96 mmol) in the form of colorless viscous oil.
- TPSCl (4.49 g, 14.82 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (2.35 g, 4.94 mmol), icosanol (2,95 g, 9,9 mmol), and 1-methylimidazole (1.17 ml, 14.82 mmol) in DCM (50 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- the organic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 34% yield (1,1 g, 1,68 mmol) in the form of colorless wax.
- TPSCl (5.54 g, 18.3 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.8 g, 6.1 mmol), hexadecyloxypropanol (1.45 g, 3.05 mmol), and 1-methylimidazole (1.45 ml, 18.3 mmol) in DCM (60 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- TPSCl (7.15 g, 23.61 mmol) is added to the mixture of monomethyl vinylphosphonate (1.95 g, 16 mmol), 4- N -benzoyl-2',3'-isopropylidenecytidine (3.05g, 7.87 mmol), and 1-methylimmidazole (1.9 ml, 23.61 mmol) in DCM (80 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- the title product is obtained by flash chromatography on silica gel using linear gradient of solvent mixture H1 (ethyl acetate:acetone:ethanol: water, volume ratios 4:1:1:1) in ethyl acetate in 52% yield (1.95 g, 4.1 mmol) as a beige foam.
- TPSCl (1.24 g, 4.08 mmol) is added to the mixture of vinylphosphonic acid from the example 7 (0.65 g, 1.36 mmol), hexadecyloxypropanol (0.81 g, 2.7 mmol), and 1-methylimidazole (1.45 ml, 18.3 mmol) in DCM (60 ml).
- the reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO 3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na 2 SO 4 .
- Organic phase is concentrated in vacuo and product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 78% yield (0.81 g, 1.06 mmol) in the form of colorless wax.
- Antimicrobial activity is assessed using the standard microdilution method determining the minimum inhibitory concentration (MIC) of the tested samples leading to inhibition of bacterial growth.
- Disposable microtitration plates are used for the tests. The samples were diluted in brain heart infusion broth (Himedia, Laboraties Pvt. Ltd., eská republika) and Mueller-Hinton broth (HiMedia Laboraties, see above) to yield a concentration range between 200 ⁇ g/ml and 1.5625 ⁇ g/ml. The plates are inoculated with a standard amount of the tested microbe - the inoculum density in each well is equal to 10 5-6 CFU/ml.
- the MIC is read after 24/48 hours of incubation at 37°C as the minimum inhibitory concentration of the tested substance that inhibits the growth of the bacterial strains.
- the minimum bactericidal concentration (MBC) is characterized as the minimum concentration of the sample required to achieve irreversible inhibition, i.e. killing the bacterium after a defined period of incubation.
- the MBC is examined by the inoculation method. With an applicator, 10 ⁇ L are transferred from microplate wells with defined concentrations of the tested sample and inoculated onto the surface of blood agar (Trios, Czech Republic). The MBC is determined as the lowest concentration that inhibited the visible growth of the used bacterium.
- Table 1 Minimum inhibitory concentration of selected novel lipophosphonoxines against a panel of reference bacterial strains MIC ⁇ g/ml Compound prepared in example E. faecalis CCM 4224 S.aureus CCM 4223 B. subtilis S.agalactiae 9 12,5 - 6,25 3,125 10 6,25 25 3,125 3,125 11 12,5 - 6,25 6,25 12 - - - 12,5 13 - - - 6,25 14 - - - 6,25 15 6,25 25-12,5 12,5-6,25 3,125 16 6.25 12,5-6,25 3,125 1,5625 17 12,5 25-12.5 6,25 6,25 Table 2 Minimum inhibitory concentration of selected novel lipophosphonoxines against a panel of resistant bacterial strains MIC ⁇ g/ml Compound prepared in example S.
- Multiresistant bacterial strains isolated from clinical isolates of patients from Teaching Hospital Olomouc MRSA - (methicilin-resistant S. aureus 4591); S. haemolyticus (fluoroquinolone-resistant strain 16568); E. faecium (vancomycin-resistant strain VanA419/ana); S. epidermidis (methicilin-resistant strain 8700/B).
- MBC minimum bactericidal concentration
- Cytotoxicity is determined after 4 hours at 37 °C in 5% CO 2 using commercial CytoTox-ONETM Homogeneous Membrane Integrity Assay (Promega, Cat.# G7892), a fluorometric method for estimating the number of non-viable cells present in multiwell plates.
- the CytoTox-ONETM Assay measures the release of lactate dehydrogenase (LDH) from the cells with a damaged membrane. LDH released into the culture medium is measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. The amount of fluorescence produced is proportional to the number of lysed cells.
- LDH lactate dehydrogenase
- Cytotoxicity of tested compounds against normal human erythroid progenitor cells was determined using commercial test CytoToxOne (Promega Corp., Madison, USA)
- Lipophosphonoxins according to this invention can be used as antibacterial agents in pharmaceutical preparations destined for the treatment of bacterial infections, components of disinfectants and/or selective growth media.
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Description
- The invention relates to novel compounds with antibacterial properties, methods of synthesis thereof, and utilisation thereof in vitro and in vivo.
- The advantages offered by antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This reduces the efficiency of antibiotic treatments and poses a serious health and economical problem. Currently, the need for novel antibiotics is becoming increasingly apparent (Davies D., Davies J., Microbiol. Mol. Biol. Rev. 2010, 74(3), 417; Kesselheim A. S., Outterson K., Health Aff. 2010, 29, 1689).
- An effective inhibitor of Giardia trophozoite growth termed phosphonoxin (1) (
Figure 1 ) with an activity that rivaled existing therapeutics was recently reported. Phosphonoxin was designed as a transient-state inhibitor of a glycosyl transferase - cyst wall synthase (CWS). CWS catalyzes synthesis of the chitin-like poly β-1-3-linked N-acetylgalactosamine [poly(GalNAc)] that comprises about 63% of the giardia cyst wall. Although phosphonoxin did not specifically inhibit cyst formation, it potently inhibited vegetative growth. (Suk D. H., Rejman D. et al., Bioorg. Med. Chem. Letters 2007, 17(10), 2811). The molecule of phosphonoxin display certain structural similarity to several types of nucleoside antibiotics: (i) polyoxins 3, (ii) muraymycins 4, and (iii) caprazamycins 5. - The polyoxins are a new group of antifungal antibiotics isolated from Streptomyces cacaoi that inhibit the growth of a number of mycelial fungi by interfering with chitin synthesis. (Isono, K., Asahi K., Suzuki S., J. Am. Chem. Soc. 1969, 91, 7490; Isono K. et al., Agricultural Biol. Chem. 1965, 29, 848; Endo A., Kakiki K., Misato J., J. Bacteriol. 1970, 104, 189; Ohta N., Kakiki K., Misato, T., Agric. Biol. Chem. 1970, 34, 1224). Polyoxin D shares a gross structural similarity with uridine diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc 2) in agreement with its role as a competitive inhibitor.
- The muraymycins, isolated from a culture broth of Streptomyces species are members of a class of naturally occurring 6'-N-alkyl-5'-β-O-aminorybosyl-C-glycyluridine antibiotics. The muraymycins bearing lipophylic side chain exhibit excelent activity against gram-positive bacteria. The muraymycins inhibit the formation of lipid II and peptidoglycan and are believed to be inhibitors of phosphor-MurNAc-pentapeptide translocase (MraY), which is responsible for the formation of lipid I in the peptidoglycan biosynthesis pathway. ( Bugg T. D. H., Lloyd A. J., Roper D. I., Infect. Dis. Drug Targets 2006, 6, 85. Kimura K., Bugg T. D. H., Nat. Prod. Rep. 2003, 20, 252; Bouhss A. et al., J. Mol. Microbiol. 1999, 34, 576; Bouhss A., et al., FEMS Microbiol. Rev. 2008, 32, 208. Bouhss, A. et al., FEMS Microbiol. Rev. 2008, 32, 208). Tanino recently described the synthesis of lipophilic muraymycin analog 6 exhibiting a significant activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). (Tanino T. et al., Med. Chem. Lett. 2010, 1, 258).
- The caprazamycins (CPZs) (5) were isolated from the culture broth of the actinomycete strain Streptomyces spp. MK730-62F2 in 2003 (Igarashi M. et al., J. Antibiot. 2003, 56, 580; Igarashi M. et al., J. Antibiot. 2005, 58, 327) and represent the most recent members of the class of naturally occurring 6'-N-alkyl-5'-β-O-aminoribosyl-C-glycyluridine antibiotics that include the liposidomycins (7) (LPMs). The CPZs have shown excellent antimycobacterial activity in vitro not only against drug-susceptible (MIC = 3.13 µg/mL) but also multidrug-resistant Mycobacterium tuberculosis strains (MIC = 3.13 µg/mL) and exhibit no significant toxicity in mice. The biological target of the 6'-N-alkyl-5'-β-O-aminoribosyl-C-glycyluridine class of antibiotics is believed to be MraY translocase (IC50= 0.05 µg/mL for LPMs). (Kimura K. et al., Agric. Biol. Chem. 1989, 53, 1811)
- This invention provides novel compound of general formula I that exhibit significant antibacterial activity, in particular against gram-positive bacteria. The advantages of these compounds include simple synthesis and modular structure allowing for further optimization of their biological properties.
-
- wherein R1 is (C8-C22)alkyl or hexadecyloxypropyl, tetradecyloxypropyl, tetradecyloxyethyl, or hexadecyloxyethyl, and
- R2 is uracil, thymin or cytosin,
- R3 is selected from the group comprising compounds of general formula II and III:
- where R4 is H or CH2OH, R5 is H or OH, R6 is H or OH, R7 is H or CH2OH, R8 is H or CH2OH, R9 is H or OH, R10 is H or OH, R11 is H or OH, R12 is H or CH2OH,
- Compounds of formula I contain several chiral centers (particularly on the phosphorus atom and in the R3 group). The presence of a chiral center allows a compound to exist as one of two possible optical isomers ((R)- or (S)-enantiomer), or as a racemic mixture of both. In this case, where several chiral centers are present, diastereoisomers and mixtures of diastereoisomers are obtained, which are all covered by general formula I and included in the scope of this invention.
- Another object of this invention is the synthesis of the novel lipophosphonoxins. The starting material for the synthesis of the final compounds arc nucleoside vinylphosphonic acids of general formula 11 (Scheme 2). These compounds are prepared by esterification of methyl vinylphosphonate 9 (Gao, Feng et al.; Chemistry-A European Journal, 2009, 15, 9, 2064 ― 2070) that is prepared according to literature procedures. The synthesis according to this invention is novel, simpler than the previously described procedures and affords good yields.
In the first step A), the comercially available dimethyl vinylphosphonate VIII (see Scheme 1) is heated overnight in 40% - 70% aqueous pyridine at 50 - 80 °C. - The obtained methyl vinylphosphonate IX is allowed to react in the second reaction step B with nucleoside X possessing free 5'-hydroxyl group and protected in the positions 2'and 3'with a group selected from 2',3'-isopropylidene, 2',3'-bis(dimethoxytrityl), 2',3'-dibenzoyl, and 2',3'-diacetyl.
- The esterification is carried out using triisopropylbenzenesulfonylchloride (TPSCl) under N-methylimidazole catalysis in a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran (THF), dichloroethane, chloroform and dichloromethane. The obtained methyl ester is purified by chromatography on silica gel using linear gradient of ethanol in chloroform. The methyl ester group is subsequently removed by heating with 40% - 70% aqueous pyridine at 50 - 80 °C overnight (Scheme 2). The obtained vinylphosphonic acid XI is purified by chromatography on silica gel using linear gradient of mixture H1 (ethyl acetate:acetone:ethanol:water, volume ratios 4:1:1:1) in ethyl acetate.
- In the next step C), the vinylphosphonic acids XI are esterified with alcohol using TPSCl as a condensing agent under N-methylimidazole catalysis in a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran (THF), dichloroethane, chloroform and dichloromethane (Scheme 3). The obtained vinylphosphonate XII is purified by chromatography on silica gel using linear gradient of ethanol in chloroform.
- The final step D) consists in Michael addition of secondary amine to the vinyl moiety of intermediate XII. The reaction is accomplished by heating in a solvent selected from the group comprising acetonitrile, dioxane, THF, dichloroethane, chloroform, ethanol, propanol, isopropanol and n-butanol at 80 - 120 °C for 4 to 12 hours. Subsequently, the protecting groups are removed from the nucleoside moiety. The final product is isolated by chromatography on silica gel using linear gradient of mixture H1 (as described above) in ethyl acetate. If required, the final product is re-purified by preparative HPLC on reverse phase.
- It is an aspect of the method of preparation of lipophosphonoxins of general formula I or their diastereomers or mixtures of such diastereomers, that heating overnight with aqueous pyridine in steps A) and/or B) is preferably carried out at 60 °C and the concentration of the aqueous pyridine solution is preferably 60 vol. %.
- In another preferred embodiment, the Michael addition in the step D is carried out in n-butanol at 105 °C; more preferably, the reaction is perfomed overnight.
- In one aspect of the present invention, the esterification in the steps B and/or C is carried out preferably in dichloromethane.
- In another preferred embodiment of the method of the preparation of lipophosphonoxins of general formula I or their diastereomers and mixtures of such diastereomers, in the step D), the nucleoside protecting groups are removed by ethanolic methylamine or aqueous ammonia: if 2',3'-protected ribonucleoside is used, the protecting group is preferably removed by treatment with 0.5 mol.l-1 hydrochloride in methanol overnight at room temperature.
- Another object of the present invention are the lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds for use as medicaments.
- Yet another object of the present invention are the lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds for use as antibacterial medicaments.
- Another object of the present invention is an antibacterial medicament containing as the active ingredient at least one lipophoshonoxin of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixture of such compounds.
- Finally, an object of the invention is the use of lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds as active components of disinfectants and/or selective growth media for in vitro cultivations.
- The compounds according to this invention possess antibacterial activity particularly against bacterial strains Enterococcus faecalis, Bacterium subtilis, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus haemolyticus, Enterococcus faecium, Staphylococcus epidermidis, including strains resistant to current antibiotics.
- The compounds according to this invention simultaneously exhibited none or very low effect on the viability of normal human erythroid progenitor cells cultivated in vitro in the range of concentrations sufficient for antibacterial activity. The same applies to cytotoxicity induced by these compounds.
- The modular structure as well as the simple synthetic procedure consisting in connecting single modules allows for large structural variations of the compounds according to this invention that can lead to the modulation of their biological activity.
-
-
Figures 1A-I depict the cell viability determined by the commercially available CellTiter-Blue® Cell Viability Assay (Promega, catalogue. no. G8082). The x-axis represents concentration of the tested compound in µg/ml in logarithmic scale, the y-axis represents cell viability in % of control. 1A: compound from the example 9, 1B: compound from the example 10, 1C: compound from the example 11, 1D: compound from the example 12, 1E: compound from the example 13, 1F: compound from the example 14, 1G: compound from the example 15, 1H: compound from the example 16, 1I: compound from the example 17. For comparison, the antibacterial activities (MIC values) of these compounds against selected bacterial species are indicated with vertical lines in the charts. -
Figures 2A-E depict the cytotoxicity determined by the commercially available CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, catalogue no. G7892). The x-axis represents concentration of the tested compound in µg/ml in logarithmic scale, the y-axis represents normalized cytotoxicity described by a dimensionless number. 2A: compound from the example 9, 2B: compound from the example 14, 2C: compound from the example 15, 2D: compound from the example 16, 2E: compound from the example 17. For comparison, the antibacterial activities (MIC values) of these compounds against selected bacterial species are indicated with vertical lines in the charts. -
- DCM
- dichloromethane
- TPSCl
- triisopropyl benzenesulfonylchloride
- IR
- infrared spectrum
- HR-ESI
- high resolution mass spectroscopy, using electrospray ionisation
- HR-EI
- high resolution mass spectroscopy, using electron impact ionisation
- n-BuOH
- n-butanol
- DMTr
- dimethoxytrityl
- THF
- tetrahydrofuran
- EC50
- half maximal effective concentration
- IC50
- half maximal inhibitory concentration
- To the mixture of the monomethyl phosphonate (9,15 g, 75 mmol), 2',3'-isopropylideneuridine (14 g, 50 mmol) and methylimidazole (5,9 ml, 150 mmol) in DCM (500 ml), TPSCl (45,43 g, 150 mmol) is added. The reaction mixture is stirred at room temperature (rt) overnight, then washed with a saturated aq NaHCO3 (2 x 500 ml) followed by washing with 3% aq citric acid (2 x 500 ml) and dried over Na2SO4. Organic phase is concentrated in vacuo and monomethyl ester of nucleosidevinylphosphonic acid is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform. The monomethyl ester intermediate in 40-70%, preferably in 60% (vol. %) aqueous pyridine (500 ml) is stirred at 50-80 °C, preferably at 60 °C overnight. The reaction mixture is concentrated in vacuo, co-evaporated in EtOH (2 x 300 ml), and dissolved in the same solvent (500 ml).
Dowex 50 in Et3N form (200 mll) is added and the suspension is stirred for 10 min. The resin is removed by filtration, and the filtrate is concentrated in vacuo. The title product is obtained by flash chromatography on silica gel using linear gradient of solvent mixture H1 (ethyl acetate:acetone:ethanol:water, volume ratios 4:1:1:1) in ethyl acetate in 48% yield (11.38 g, 23.93 mmol ) as a yellowish foam. - 1H NMR (499.8 MHz, CD3OD): 1.35, 1.54 (2 × q, 2 × 3H, 4 J = 0.5, (CH3)2C); 3.98 (dd, 2H, J H,P = 5.3, J 5',4' = 3.4, H-5'); 4.35 (m, 1H, H-4'); 4.90 (m, 2H, H-2',3'); 5.74 (d, 1H, J 5,6 = 8.1, H-5); 5.86 (ddd, 1H, J H,P = 46.0, J cis = 12.3, J gem = 3.1, CHcis Htrans =CHP); 5.95 (d, 1H, J 1',2' = 2.5, H-1'); 6.00 (ddd, 1H, J H,P = 22.8, J trans = 18.7, J gem = 3.1, CHcis Htrans=CHP); 6.12 (ddd, 1H, J H,P = 19.7, J trans = 18.7, J cis = 12.3, =CHP); 7.86 (d, 1H, J 6,5 = 8.1, H-6).
- 13C NMR (125.7 MHz, CD3OD): 25.53, 27.55 ((CH3)2C); 65.31 (d, J C,P = 4.8, CH2-5'); 82.49 (CH-3'); 85.67 (CH-2'); 86.56 (d, J C,P = 8.0, CH-4'); 93.40 (CH-1'); 102.98 (CH-5); 114.99 (C(CH3)2); 130.74 (CH2=CHP); 132.70 (d, J C,P = 174.3, =CHP); 143.42 (CH-6); 152.16 (C-2); 166.15 (C-4).
- 31P NMR (202.3 MHz, CD3OD): 13.16.
- IR νmax(KBr) 3200 (w, br), 2977 (m), 2939 (m), 2803-2100 (s-vw), 1715 (s, sh), 1695 (vs), 1630 (w, sh), 1612 (w, sh), 1469 (m), 1434 (m), 1398 (m), 1384 (m), 1373 (m, sh), 1273 (m), 1214 (s), 1115 (m, sh), 1070 (s, br), 1037 (s), 1018 (m, sh), 995 (w), 765 (w), 517 (w) cm-1.
- HR-ESI C14H18O8N2P (M-H)- calcd 373.0806; found 373.0809
-
- TPSCl (4.68 g, 15.45 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.93 g, 5.15 mmol), tetradekanol (2.2 g, 10.31 mmol), and N-methylimidazole (1.22 ml, 15.45 mmol) in DCM (50 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. The organic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 40% yield (1.17 g, 2.05 mmol) in the form of yellowish viscous oil.
A mixture of diastereoisomers ~ 6:5 - 1H NMR (499.8 MHz, CDCl3): 0.88 (m, 6H, CH3 (CH2)13); 1.20-1.35 (m, 44H, CH3(CH2 )11CH2CH2-O); 1.350, 1.354, 1.573, 1.576 (4 × q, 4 × 3H, 4 J = 0.7, (CH3)2C); 1.67 (m, 4H, CH3(CH2)11 CH 2CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, J H,P = 7.3, J vic = 6.7, CH3(CH2)11CH2 CH2O); 4.19-4.29 (m, 4H, H-5'); 4.35-4.39 (m, 2H, H-4'); 4.85, 4.86 (2 × dd, 2 × 1H, J 3',2' = 6.4, J 3',4' = 3.6, H-3'); 4.88, 4.89 (2 × dd, 2 × 1H, J 2',3' = 6.4, J 2',1' = 2.3, H-2'); 5.71, 5.72 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.77, 5.81 (2 × d, 2 × 1H, J 1',2' = 2.3, H-1'); 6.03, 6.04 (2 × ddd, 2 × 1H, J H,P = 22.9, J trans = 18.6, J cis = 12.7, =CHP); 6.16, 6.19 (2 × ddd, 2 × 1H, J H,P = 51.6, J cis = 12.7, J gem = 2.0, CHcis Htrans =CHP); 6.33, 6.34 (2 × ddd, 2 × 1H, J H,P = 25.7, J trans = 18.7, J gem = 2.0, CHcis Htrans=CHP); 7.38, 7.43 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6); 9.34 (bs, 2H, NH).
13 C NMR (125.7 MHz, CDCl3): 14.08 (CH3(CH2)13); 22.64 (CH3(CH2)11CH2CH2O); 25.23, 25.25 ((CH3)2C); 25.43 (CH3(CH2)11CH2CH2O); 27.08, 27.09 ((CH3)2C); 29.10, 29.30, 29.46, 29.52, 29.59, 29.60, 29.62, 29.64 (CH3(CH2)11CH2CH2O); 30.39 (d, J C,P = 6.2, CH3(CH2)11CH2CH2O); 31.87 (CH3(CH2)11CH2CH2O); 64.93, 65.01 (d, J C,P = 5.5, CH2-5'); 66.47, 66.48 (d, J C,P = 5.7, CH3(CH2)11CH2CH2O); 80.57, 80.66 (CH-3'); 84.45, 84.53 (CH-2'); 85.26, 85.55 (d, J C,P = 7.0, CH-4'); 93.43, 93.83 (CH-1'); 102.49, 102.58 (CH-5); 114.54, 114.59 (C(CH3)2); 124.97, 125.02 (d, J C,P = 184.3, =CHP); 136.60, 136.62 (CH2=CHP); 141.47, 141.55 (CH-6); 149.96, 149.99 (C-2); 163.11 (C-4).
31P NMR (202.3 MHz, CDCl3): 18.59, 18.74.
IR v max(CHCl3) 3390 (w), 3170 (w, br), 2957 (s), 2928 (vs), 2856 (s), 1715 (vs), 1695 (vs), 1634 (m), 1616 (m), 1457 (s), 1421 (m), 1399 (m), 1384 (s), 1378 (s), 1240 (vs, br), 1121 (s), 1109 (s), 1070 (vs), 1011 (vs), 988 (s), 973 (s, sh), 860 (s), 544 (m), 512 (m) cm-1.
HR-ESI C28H48O8N2P [M+H]+ calcd 571.31428, found 571.31445, C28H47O8N2NaP [M+Na]+ calcd 593.29622, found 593.29616. -
- TPSCI (4.12 g, 13.62 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.7 g, 4.54 mmol), hexadecanol (2.9 g, 12 mmol), and 1-methylimidazole (1.08 ml, 13.62 mmol) in DCM (50 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. The organic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 50% yield (1.38 g, 2.3 mmol) in the form of colorless viscous oil.
- 1H NMR (499.8 MHz, CDCl3): 0.88 (m, 6H, CH3 (CH2)15); 1.22-1.35 (m, 52H, CH3(CH2 )13CH2CH2-O); 1.350, 1.354, 1.573, 1.577 (4 × q, 4 × 3H, 4 J = 0.7, (CH3)2C); 1.67 (m, 4H, CH3(CH2)13CH2 CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, J H,P = 7.3, J vic = 6.7, CH3(CH2)13CH2CH2 O); 4.19-4.30 (m, 4H, H-5'); 4.35-4.39 (m, 2H, H-4'); 4.85, 4.86 (2 × dd, 2 × 1H, J 3',2' = 6.4, J 3',4' = 3.6, H-3'); 4.878, 4.8984 (2 × dd, 2 × 1H, J 2',3' = 6.4, J 2',1' = 2.3, H-2'); 5.71, 5.72 (2 × dd, 2 × 1H, J 5,6 = 8.1, J 5,NH = 2.2, H-5); 5.78, 5.81 (2 × d, 2 × 1H, J 1',2' = 2.3, H-1'); 6.03, 6.05 (2 × ddd, 2 × 1H, J H,P = 22.8, J trans = 18.6, J cis = 12.8, =CHP); 6.12, 6.13 (2 × ddd, 2 × 1H, J H,P = 51.7, J cis = 12.8, J gem = 2.0, CHcis Htrans =CHP); 6.33, 6.34 (2 × ddd, 2 × 1H, J H,P = 24.8, J trans = 18.6, J gem = 2.0, CHcis Htrans=CHP); 7.38, 7.43 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6); 9.15, 9.18 (2 × bs, 2 × 2H, NH).
- 13C NMR (125.7 MHz, CDCl3): 14.08 (CH3(CH2)15); 22.65 (CH3(CH2)13CH2CH2O); 25.23, 25.26 ((CH3)2C); 25.43 (CH3(CH2)13CH,CH2O); 27.08, 27.10 ((CH3)2C); 29.11, 29.32, 29.47, 29.53, 29.60, 29.61, 29.63, 29.65 (CH3(CH2)13CH2CH2O); 30.40 (d, J C,P = 6.3, CH3(CH2)13CH2CH2O); 31.88 (CH3(CH2)13CH2CH2O); 64.93, 65.00 (d, J C,P = 5.5, CH2-5'); 66.47, 66.48 (d, J C,P = 5.7, CH3(CH2)13CH2CH2O); 80.57, 80.66 (CH-3'); 84.46, 84.54 (CH-2'); 85.25, 85.55 (d, J C,P = 7.1, CH-4'); 93.40, 93.80 (CH-1'); 102.49, 102.57 (CH-5); 114.55, 114.60 (C(CH3)2); 124.99, 125.04 (d, J C,P 184.2, =CHP); 136.62, 136.66 (d, J C,P = 2.0, CH2=CHP); 141.46, 141.55 (CH-6); 149.92, 149.95 (C-2); 163.05, 163.07 (C-4).
- 31P NMR (202.3 MHz, CDCl3): 18.59, 18.73.
- HR-ESI C30H51O8N2PNa (M+Na)+ calcd 621.32752; found 621.32778
-
- TPSCl (4.16 g, 13.74 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.7 g, 4.54 mmol), octadecanol (2.48 g, 9.17 mmol), and 1-methylimidazole (1.08 ml, 13.74 mmol) in DCM (50 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. The rganic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 43% yield (1,23 g, 1,96 mmol) in the form of colorless viscous oil.
- 1H NMR (499.8 MHz, CDCl3): 0.88 (m, 6H, CH3 (CH2)17); 1.20-1.40 (m, 60H, CH3(CH2 )15CH2CH2-O); 1.350, 1.354, 1.573, 1.576 (4 × q, 4 × 3H, 4 J = 0.7, (CH3)2C); 1.66 (m, 4H, CH3(CH2)15CH2 CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, J H,P = 7.3, J vic = 6.7, CH3(CH2)15CH2CH2 O); 4.19-4.29 (m, 4H, H-5'); 4.34-4.39 (m, 2H, H-4'); 4.85, 4.86 (2 × dd, 2 × 1H, J 3',2' = 6.4, J 3',4' = 3.7, H-3'); 4.88, 4.89 (2 × dd, 2 × 1H, J 2',3' = 6.4, J 2',1' = 2.3, H-2'); 5.71, 5.72 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.77, 5.81 (2 × d, 2 × 1H, J 1',2' = 2.3, H-1'); 6.03, 6.04 (2 × ddd, 2 × 1H, J H,P = 22.9, J trans = 18.6, J cis = 12.7, =CHP); 6.16, 6.19 (2 × ddd, 2 × 1H, J H,P = 51.7, J cis = 12.7, J gem = 2.0, CHcis Htrans =CHP); 6.33, 6.34 (2 × ddd, 2 × 1H, J H,P = 25.6, J trans = 18.6, J gem = 2.0, CHcis Htrans=CHP); 7.38, 7.43 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6); 9.37 (bs, 2H, NH).
- 13C NMR (125.7 MHz, CDCl3): 14.08 (CH3(CH2)17); 22.64 (CH3(CH2)15CH2CH2O); 25.22, 25.25 ((CH3)2C); 25.43 (CH3(CH2)15CH2CH2O); 27.07, 27.09 ((CH3)2C); 29.10, 29.31, 29.46, 29.53, 29.60, 29.61, 29.63, 29.65 (CH3(CH2)15CH2CH2O); 30.39 (d, J C,P = 6.2, CH3(CH2)15CH2CH2O); 31.87 (CH3(CH2)15CH2CH2O); 64.93, 65.01 (d, J C,P = 5.5, CH2-5'); 66.46, 66.47 (d, J C,P = 5.7, CH3(CH2)15CH2CH2O); 80.57, 80.66 (CH-3'); 84.45, 84.53 (CH-2'); 85.27, 85.56 (d, J C,P = 7.0, CH-4'); 93.44, 93.83 (CH-1'); 102.49, 102.58 (CH-5); 114.53, 114.58 (C(CH3)2); 124.97, 125.01 (d, J C,P = 184.2, =CHP); 136.62, 136.66 (d, J C,P = 2.0, CH2=CHP); 141.47, 141.56 (CH-6); 149.97, 150.00 (C-2); 163.14, 163.16 (C-4).
- 31P NMR (202.3 MHz, CDCl3): 18.59, 18.74.
- IR v max(CHCl3) 3390 (w), 3170 (w, br), 2928 (vs), 2855 (vs), 1716 (vs), 1695 (vs), 1634 (m), 1615 (w), 1457 (s), 1420 (m), 1399 (m), 1385 (s), 1378 (s), 1246 (s, br), 1121 (s), 1110 (s), 1071 (s), 1010 (s), 988 (s), 974 (s, sh), 860 (m), 544 (w), 512 (w) cm-1.
- HR-ESI C32 H56 O8 N2 P [M+H]+ calcd 627.37688, found 627.37738.
-
- TPSCl (4.49 g, 14.82 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (2.35 g, 4.94 mmol), icosanol (2,95 g, 9,9 mmol), and 1-methylimidazole (1.17 ml, 14.82 mmol) in DCM (50 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. The organic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 34% yield (1,1 g, 1,68 mmol) in the form of colorless wax.
- 1H NMR (499.8 MHz, CDCl3): 0.88 (m, 6H, CH3 (CH2)19); 1.20-1.36 (m, 68H, CH3(CH2 )17CH2CH2-O); 1.351, 1.354, 1.574, 1.577 (4 × q, 4 × 3H, 4 J = 0.7, (CH3)2C); 1.66 (m, 4H, CH3(CH2)17CH2 CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, J H,P = 7.3, J vic = 6.7, CH3(CH2)17CH2CH2 O); 4.19-4.29 (m, 4H, H-5'); 4.35-4.39 (m, 2H, H-4'); 4.84, 4.86 (2 × dd, 2 × 1H, J 3',2' = 6.4, J 3',4' = 3.4, H-3'); 4.87, 4.89 (2 × dd, 2 × 1H, J 2',3' = 6.4, J 2',1' = 2.3, H-2'); 5.706, 5.714 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.77, 5.80 (2 × d, 2 × 1H, J 1',2' = 2.3, H-1'); 6.03, 6.06 (2 × ddd, 2 × 1H, J H,P = 22.8, J trans = 18.6, J cis = 12.7, =CHP); 6.17, 6.19 (2 × ddd, 2 × 1H, J H,P = 51.7, J cis = 12.7, J gem = 2.0, CHcis Htrans =CHP); 6.33, 6.34 (2 × ddd, 2 × 1H, J H,P = 25.6, J trans = 18.6, J gem = 2.0, CHcis Htrans=CHP); 7.37, 7.43 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6); 9.08 (bs, 2H, NH).
- 13C NMR (125.7 MHz, CDCl3): 14.10 (CH3(CH2)19); 22.66 (CH3(CH2)17CH2CH2O); 25.24, 25.26 ((CH3)2C); 25.44 (CH3(CH2)17CH2CH2O); 27.09, 27.11 ((CH3)2C); 29.12, 29.33, 29.48, 29.55, 29.62, 29.65, 29.67 (CH3(CH2)17CH2CH2O); 30.41 (d, J C,P = 6.3, CH3(CH2)17CH2CH2O); 31.89 (CH3(CH2)17CH2CH2O); 64.92, 64.99 (d, J C,P = 5.5, CH2-5'); 66.48, 66.50 (d, J C,P = 5.7, CH3(CH2)17CH2CH2O); 80.57, 80.66 (CH-3'); 84.47, 84.56 (CH-2'); 85.25, 85.54 (d, J C,P = 7.0, CH-4'); 93.44, 93.83 (CH-1'); 102.50, 102.59 (CH-5); 114.56, 114.62 (C(CH3)2); 124.98, 125.02 (d, J C,P = 184.2, =CHP); 136.65, 136.70 (d, J C,P = 1.9, CH2=CHP); 141.41, 141.50 (CH-6); 149.90, 149.93 (C-2); 162.92, 162.95 (C-4).
- 31P NMR (202.3 MHz, CDCl3): 18.59, 18.74.
- IR v max(CHCl3) 3389 (w), 3167 (w, vbr), 2928 (vs), 2855 (s), 1715 (vs), 1695 (vs), 1634 (m), 1615 (w), 1457 (s), 1420 (w), 1399 (m), 1384 (s), 1378 (s), 1249 (s, sh), 1122 (m), 1110 (s), 1078 (s), 1011 (s), 989 (s), 975 (s, sh), 860 (m), 544 (w), 512 (w) cm-1.
- HR-ESI C34H60O8N2P [M+H] calcd 655.40818, found 655.40857, C34H59O8N2NaP [M+Na] calcd 677.39012, found 677.39032.
-
- TPSCl (5.54 g, 18.3 mmol) is added to the mixture of vinylphosphonic acid from the example 1 (1.8 g, 6.1 mmol), hexadecyloxypropanol (1.45 g, 3.05 mmol), and 1-methylimidazole (1.45 ml, 18.3 mmol) in DCM (60 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. The organic phase is concentrated in vacuo and the product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 71% yield (1.42 g, 2.16 mmol) in the form of colorless wax.
A mixture of diastereoisomers ~ 1:1
1H NMR (500.0 MHz, CDCl3): 0.88 (m, 6H, CH3 (CH2)15); 1.23-1.33 (m, 52H, CH3(CH2 )13CH2CH2-O); 1.348, 1.352 (2 × q, 2 × 3H, 4 J= 0.6, (CH3)2C); 1.54 (m, 4H, CH3(CH2 )13CH2CH2O); 1.57 (s, 6H, (CH3)2C); 1.92, 1.94 (2 × p, 2 × 2H, J vic = 6.1, OCH2CH2 CH2OC16H33); 3.38, 3.39 (2 × t, 2 × 2H, J vic, = 6.7, CH3(CH2)13CH2 CH2O); 3.48, 3.49 (2 × t, 2 × 2H, J vic = 6.1, OCH2CH2CH2OC16H33); 4.13, 4.15 (2 × td, 2 × 2H, J vic = 6.1, J H,P = 4.8, OCH2 CH2CH2OC16H33); 4.20-4.30 (m, 4H, H-5'); 4.34-4.38 (m, 2H, H-4'); 4.85, 4.86 (2 × dd, 2 × 1H, J 3',2' = 6.5, J 3',4' = 3.6, H-3'); 4.92 (dd, 2H, J 2',3' = 6.5, J 2',1' = 2.3, H-2'); 5.700, 5.704 (2 × d, 2 × 1H, J 5,6 = 8.1 H-5); 5.74, 5.76 (2 × d, 2 × 1H, J 1',2' = 2.3, H-1'); 6.03, 6.05 (2 × ddd, 2 × 1H, J H,P = 22.9, J trans = 18.4, J cis = 12.7, =CHP); 6.16, 6.18 (2 × ddd, 2 × 1H, J H,P = 51.8, J cis = 12.7, J gem = 1.9, CHcis Htrans =CHP); 6.33, 6.35 (2 × ddd, 2 × 1H, J H,P = 25.5, J trans = 18.4, J gem = 1.9, CHcis Htrans=CHP); 7.34, 7.39 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13 C NMR (125.7 MHz, CDCl3): 14.07 (CH3(CH2)15); 22.62 (CH3(CH2)13CH2CH2O); 25.19, 25.21 ((CH3)2C); 26.08 (CH3(CH2)14CH2O); 27.04, 27.06 ((CH3)2C); 29.29, 29.45, 29.55, 29.57, 29.58, 29.63 (CH3(CH2)14CH2O); 30.66, 30.68 (d, J C,P = 6.4, OCH2CH2CH2OC16H33); 31.85 (CH3(CH2)13CH2CH2O); 63.56 63.57 (d, J C,P = 5.5, OCH2CH2CH2OC16H33); 64.92, 65.01 (d, J C,P = 5.5, CH2-5'); 66.32, 66.36 (OCH2CH2CH2OC16H33); 71.15, 71.16 (CH3(CH2)14CH2O); 80.60, 80.67 (CH-3'); 84.44, 84.53 (CH-2'); 85.38, 85.62 (d, J C,P = 7.1, CH-4'); 93.82, 94.12 (CH-1'); 102.57, 102.64 (CH-5); 114.42, 114.46 (C(CH3)2); 124.75, 124.80 (d, J C,P = 184.0, =CHP); 136.75, 136.77 (d, J C,P = 1.9, CH2=CHP); 141.43, 141.48 (CH-6); 150.16 (C-2); 163.29, 163.32 (C-4).
31 P NMR (202.3 MHz, CDCl3): 18.64, 18.80.
IR vmax(KBr) 2925 (vs), 2854 (s), 1709 9vs, sh), 1696 (vs), 1630 (w), 1459 (m), 1421 (m), 1400 (w, sh), 1381 (m), 1270 (m, sh), 1250 (m, sh), 1109 (s), 1078 (s), 1028 (s), 1012 (s), 972 (m, sh), 859 (m), 762 (w), 548 (w), 514 (w) cm-1.
HR-ESI C33H58O9N2P (M+H)+ calcd 657.3874; found 657.3876 -
- TPSCl (7.15 g, 23.61 mmol) is added to the mixture of monomethyl vinylphosphonate (1.95 g, 16 mmol), 4-N-benzoyl-2',3'-isopropylidenecytidine (3.05g, 7.87 mmol), and 1-methylimmidazole (1.9 ml, 23.61 mmol) in DCM (80 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. Organic phase is concentrated in vacuo and monomethyl intermediate obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform is without detailed characterisation stirred in 40-70%, preferably in 60% (vol. %) aqueous pyridine (100 ml) at 50-80 °C, preferably at 60 °C, overnight. The reaction mixture is concentrated in vacuo, co-evaporated in EtOH (2 x 100 ml), and dissolved in the same solvent (100 ml).
Dowex 50 in Et3N form (80 ml) is added and the suspension is stirred for 10 min. The resin is removed by filtration, and the filtrate is concentrated in vacuo. The title product is obtained by flash chromatography on silica gel using linear gradient of solvent mixture H1 (ethyl acetate:acetone:ethanol: water, volume ratios 4:1:1:1) in ethyl acetate in 52% yield (1.95 g, 4.1 mmol) as a beige foam. NMR of 9b has shown Et3NH+ salt (not free acid):
1 H NMR (600.1 MHz, CD3OD): 1.31 (t, 9H, J vic = 7.3, CH3 CH2N); 1.37, 1.57 (2 × q, 2 × 3H, 4 J = 0.7, (CH3)2C); 3.20 (q, 6H, J vic = 7.3, CH3CH2 N); 4.02 (ddd, 1H, J gem = 11.5, J H,P = 6.0, J 5'b,4' = 3.4, H-5'b); 4.06 (ddd, 1H, J gem = 11.5, J H,P = 4.5, J 5'a,4' = 3.0, H-5'a); 4.50 (m, 1H, H-4'); 4.91 (dd, 1H, J 3',2' = 6.0, J 3',4' = 2.2, H-3'); 4.93 (dd, 1H, J 2',3' = 6.0, J 2',1' = 2.3, H-2'); 5.86 (ddd, 1H, J H,P = 46.0, J cis = 12.4, J gem = 3.1, CHcis Htrans =CHP); 6.00 (d, 1H, J 1',2' = 2.3, H-1'); 6.01 (ddd, 1H, J H,P = 22.9, J trans = 18.7, J gem = 3.1, CHcis Htrans=CHP); 6.11 (ddd, 1H, J H,P = 19.6, J trans = 18.7, J cis = 12.4, =CHP); 7.55 (m, 2H, H-m-Ph); 7.62 (bd, 1H, J 5,6 = 7.5, H-5) 7.64 (m, 1H, H-p-Ph); 7.98 (m, 2H, H-o-Ph); 8.35 (d, 1H, J 6,5 = 7.5, H-6).
13C NMR (150.9 MHz, CD3OD): 9.18 (CH3CH2N); 25.45, 27.47 ((CH3)2C); 47.73 (CH3CH2N); 65.09 (d, J C,P = 4.8, CH2-5'); 82.47 (CH-3'); 87.16 (CH-2'); 87.91 (d, J C,P = 8.1, CH-4'); 95.59 (CH-1'); 98.54 (CH-5); 114.69 (C(CH3)2); 129.16 (CH-o-Ph); 129.82 (CH-m-Ph); 130.80 (CH2=CHP); 132.71 (d, J C,P = 174.8, =CHP); 134.05 (CH-p-Ph); 134.78 (C-i-Ph); 147.24 (CH-6); 157.90 (C-2); 164.99 (C-4); 169.02 (CO).
31 P NMR (202.3 MHz, CD3OD): 13.02.
HR-ESI C21H23O8N3P (M-H)+ calcd 476.1223; found 476.1221. -
- TPSCl (1.24 g, 4.08 mmol) is added to the mixture of vinylphosphonic acid from the example 7 (0.65 g, 1.36 mmol), hexadecyloxypropanol (0.81 g, 2.7 mmol), and 1-methylimidazole (1.45 ml, 18.3 mmol) in DCM (60 ml). The reaction mixture is stirred overnight at rt, then extracted/washed with saturated solution of NaHCO3 (2 x 100 ml), 3% aqueous citric acid (2 x 100 ml) and dried over Na2SO4. Organic phase is concentrated in vacuo and product is obtained by flash chromatography on silica gel using linear gradient of ethanol in chloroform in 78% yield (0.81 g, 1.06 mmol) in the form of colorless wax.
- 1H NMR (600.1 MHz, CD3OD): 0.895 (m, 6H, CH3 (CH2)15); 1.24-1.35 (m, 52H, CH3(CH2 )13CH2CH2O); 1.368, 1.371 (2 × q, 2 × 3H, 4 J = 0.6, (CH3)2C); 1.51 (m, 4H, CH3(CH2)13CH2 CH2O); 1.57 (s, 6H, (CH3)2C); 1.89, 1.91 (2 × p, 2 × 2H, J vic = 6.1, OCH2CH2 CH2OC16H33); 3.38, 3.39 (2 × t, 2 × 2H, J vic = 6.7, CH3(CH2)13CH2CH2 O); 3.47 (td, 2H, J vic = 6.1, J H,P = 1.2, OCH2CH2CH2 OC16H33); 3.49 (t, 2H, J vic = 6.1, OCH2CH2CH2 OC16H33); 4.09-4.16 (m, 4H, OCH2 CH2CH2OC16H33); 4.27-4.36 (m, 4H, H-5'); 4.48 (m, 2H, H-4'); 4.91 (dd, 2H, J 3',2' = 6.2, J 3',4' = 3.6, H-3'); 5.06, 507 (2 × dd, 2 × 1H, J 2',3' = 6.2, J 2',1' = 1.8, H-2'); 5.87, 5.88 (2 × d, 2 × 1H, J 1',2' = 1.8, H-1'); 6.145, 6.17 (2 × ddd, 2 × 1H, J H,P = 24.0, J trans = 18.4, J cis = 12.8, =CHP); 6.257, 6.261 (2 × ddd, 2 × 1H, J H,P = 52.0, J cis = 12.8, J gem = 2.1, CHcis Htrans =CHP); 6.30, 6.31 (2 × ddd, 2 × 1H, J H,P = 26.0, J trans = 18.4, J gem = 2.1, CHcis Htrans=CHP); 7.54 (m, 4H, H-m-Bz); 7.59 (d, 2H, J 5,6 = 7.7, H-5); 7.64 (m, 2H, H-p-Bz); 7.99 (m, 4H, H-o-Bz); 8.16, 8.17 (2 × d, 2 × 1H, J 6,5 = 7.7, H-5).
- 13C NMR (150.9 MHz, CD3OD): 14.48 (CH3(CH2)15); 23.75 (CH3(CH2)13CH2CH2O); 25.46, 25.48 ((CH3)2C); 27.27 (CH3(CH2)14CH2O); 27.43 ((CH3)2C); 30.49, 30.61, 30.78, 30.80, 30.81 (CH3(CH2)14CH2O); 31.70 (d, J C,P = 6.5, OCH2 CH2CH2OC16H33); 33.09 (CH3(CH2)13CH2CH2O); 64.96, 64.98 (d, J C,P = 5.7, OCH2CH2CH2OC16H33); 66.97, 67.04 (d, J C,P = 5.6, CH2-5'); 67.42, 67.43 (OCH2CH2CH2OC16H33); 72.10 (CH3(CH2)14CH2O); 82.64, 82.635, 82.644 (CH-3'); 86.57, 86.58 (CH-2'); 88.23, 88.30 (d, J C,P = 7.2, CH-4'); 97.60, 97.71 (CH-1'); 98.51, 98.54 (CH-5); 115.10, 115.11 (C(CH3)2); 125.63, 125.67 (d, J C,P = 183.9, =CHP); 129.25 (CH-o-Bz); 129.83 (CH-m-Bz); 134.14 (CH-p-Bz); 134.70 (C-i-Bz); 138.01, 138.04 (d, J C,P = 1.9, CH2=CHP); 148.26, 148.34 (CH-6); 157.58, 157.60 (C-2); 165.58 (C-4); 169.19 (CO-Bz).
- 31 P NMR (202.3 MHz, CD3OD): 19.62, 19.72.
IR v max(CHCl3) 3406 (w), 2928 (s), 2856 (m), 1703 (m), 1670 (s), 1628 (m), 1603 (w), 1554 (m), 1582 (w), 1497 (m, sh), 1480 (vs), 1455 (w, sh), 1400 (m), 1385 (m), 1377 (m), 1301 (m), 1248 (s, sh), 1186 (w), 1158 (m), 1121 (m), 1110 (m), 1078 (s), 1070 (s), 1028 (m), 1003 (m, sh), 989 (m), 909 (w), 865 (w), 842 (vw), 708 (w), 687 (w), 512 (w) cm-1.
HR-EI C40H62N3O9P (M+H)+ calcd 759.4224; found 759.4225 -
- The mixture of (3R,4R)-3,4-dihydroxypyrrolidine (0.12 g, 1.18 mmol) and vinylphosphonate from the example 6 (0.4 g, 0.61 mmol) in n-BuOH (15 ml) is stirred at 105 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. This compound is dissolved in 0.5 M methanolic HCl (30 ml) and the mixture is stirred at rt for 4 h. The final product is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate in in 34% overall yield (152 mg, 0.205 mmol) and lyophilized from water. A mixture of diastereoisomers ∼ 6:5
1H NMR (499.8 MHz, CD3OD): 0.90 (m, 6H, CH 3(CH2)15); 1.25-1.38 (m, 52H, CH3(CH 2)13CH2CH2-O); 1.55 (m, 4H, CH3(CH2)13CH2 CH2O); 1.93 (m, 4H, OCH2CH2 CH2OC16H33); 2.12-2.25 (m, 4H, CH2P); 2.71 (dd, 4H, J gem = 10.6, J vic = 3.0, H-2b,5b-pyrr); 2.86-3.00 (m, 4H, CH2N); 3.116, 3.118 (2 × dd, 2 × 2H, J gem = 10.6, J vic = 5.0, H-2a,5a-pyrr); 3.42, 3.43 (2 × t, 2 × 2H, J vic = 6.6, CH3(CH2)13CH2CH2 O); 3.515, 3.522 (2 × t, 2 × 2H, J vic = 6.1, OCH2CH2CH2 OC16H33); 4.08 (m, 4H, OCH2 CH2CH2OC16H33); 4.11-4.21 (m, 8H, H-3',4', H-3,4-pyrr); 4.216, 4.218 (2 × dd, 2 × 1H, J 2',3' = 5.1, J 2',1' = 4.1, H-2'); 4.26 (ddd, 1H, J gem = 11.6, J H,P = 6.8, J 5'b,4'= 4.5, H-5'b); 4.30-4.34 (m, 2H, H-5'); 4.36 (ddd, 1H, J gem = 11.6, J H,P = 6.6, J 5'a,4' = 2.9, H-5'a); 5.751, 5.753 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.845 (d, 2H, J 1',2'= 4.1, H-1'); 7.70, 7.73 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (125.7 MHz, CD3OD): 14.47 (CH3(CH2)15); 23.73 (CH3(CH2)14CH2O); 24.75, 24.81 (d, J C,P = 139.6, CH2P); 27.28, 30.47, 30.63, 30.76, 30.79 (CH3(CH2)14CH2O); 31.75, 31.78 (d, J C,P = 6.2, OCH2CH2CH2OC16H33); 33.06 (CH3(CH2)14CH2O); 50.81, 50.85 (CH2N); 60.95, 60.98 (CH2-2,5-pyrr); 64.86, 64.93 (d, J C,P = 6.7, OCH2CH2CH2OC16H33); 66.52, 66.54 (d, J C,P = 6.2, CH2-5'); 67.49, 67.53 (OCH2CH2CH2OC16H33); 70.79, 70.86 (CH-3'); 72.14 (CH3(CH2)14CH2O); 74.87, 74.93 (CH-2'); 78.12, 78.19 (CH-3,4-pyrr); 83.57, 83.58 (d, J C,P = 6.6, CH-4'); 91.75, 91.85 (CH-1'); 103.02 (CH-5); 142.58, 142.64 (CH-6); 152.18, 152.20 (C-2); 165.99 (C-4).
31P NMR (202.3 MHz, CD3OD): 31.37, 31.64.
IR vmax(KBr) 3405 (s, vbr), 3063 (w), 2960 (m, sh), 2925 (vs), 2854 (s), 2810 (w, sh), 1696 (vs), 1629 (w, sh), 1466 (m), 1460 (m, sh), 1418 (w), 1384 (m), 1266 (m), 1223 (m), 1111 (s), 1075 (m, sh), 1046 (s, br), 998 (m), 821 (w), 765 (w), 721 (w) cm-1.
HR-ESI C34H63O11N3P (M+H)+ calcd 720.41947; found 720.41939. -
- The mixture of (3R,4R)-3,4-dihydroxypyrrolidine (0.05 g, 0.49 mmol) and benzoylcytosine vinylphosphonate from example 9 (0.34 g, 0.45 mmol) in n-BuOH (10 ml) is stirred at 100 °C overnight. The solvent is removed and the protected intermediate is purified by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. White foam obtained after evaporation of solvents is without further characterization (only LC-MS confirmation) dissolved in 8M ethanolic methylamine (10 ml) and stirred at rt overnight. The mixture is concentrated in vacuo, co-evaporated with ethanol (2x 20 ml), dissolved in 0.2M methanolic HCl (10 ml), and the reaction mixture is stirred at rt for 4 hours. After concentration in vacuo the final product is obtained by preparative HPLC in 27% overall yield (90.6 mg, 120 µM) after lyofilisation from water.
A mixture of diastereoisomers ∼ 8:7
1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H, CH 3(CH2)15); 1.25-1.38 (m, 52H, CH3(CH 2)13CH2CH2-O); 1.55 (m, 4H, CH3(CH2)13CH2 CH2O); 1.93 (m, 4H, OCH2CH2 CH2OC16H33); 2.10-2.18 (m, 4H, CH2P); 2.56 (bm, 4H, H-2b,5b-pyrr); 2.75-2.87 (bm, 4H, CH2N); 3.00 (bm, 4H, H-2a,5a-pyrr); 3.41, 3.43 (2 × t, 2 × 2H, J vic = 6.7, CH3(CH2)13CH2CH2 O); 3.51, 3.53 (2 × t, 2 × 2H, J vic = 6.0, OCH2CH2CH2 OC16H33); 4.04 (m, 4H, H-3,4-pyrr); 4.08-4.23 (m, 10H, H-2',3',4'-pyrr, OCH2 CH2CH2OC16H33); 4.25-4.42 (m, 4H, H-5'); 5.83, 5.84 (d, 2H, J 1',2' = 3.0, H-1'); 5.93, 5.94 (2 × d, 2 × 1H, J 5,6 = 7.6, H-5); 7.77, 7.79 (2 × d, 2 × 1H, J 6,5 = 7.6, H-6).
13C NMR (150.9 MHz, CD3OD): 14.47 (CH3(CH2)15); 23.75 (CH3(CH2)14CH2O); 25.09, 25.14 (d, J C,P = 139.1, CH2P); 27.29, 27.30, 30.49, 30.64, 30.77, 30.80 (CH3(CH2)14CH2O); 31.76, 31.79 (d, J C,P = 6.3, OCH2CH2CH2OC16H33); 33.08 (CH3(CH2)14CH2O); 50.55 (CH2N); 60.99, 61.02 (CH2-2,5-pyrr); 64.71, 64.79 (d, J C,P = 6.6, OCH2CH2CH2OC16H33); 66.24, 66.31 (d, J C,P = 6.4, CH2-5'); 67.51, 67.55 (OCH2CH2CH2OC16H33); 70.52, 70.55 (CH-3'); 72.15 (CH3(CH2)14CH2O); 75.68, 75.70 (CH-2'); 78.61, 78.66 (CH-3,4-pyrr); 83.13, 83.16 (d, J C,P = 6.6, CH-4'); 92.89, 92.90 (CH-1'); 96.17, 96.18 (C-5); 142.56, 142.64 (CH-6); 158.26, 158.27 (C-2); 167.646, 167.653 (C-4).
31P NMR (202.3 MHz, CD3OD): 32.67, 32.99.
IR v max(CHCl3) 3341 (m, vbr), 3220 (m, vbr), 2927 (vs), 2855 (s), 1649 (vs), 1609 (m, sh), 1578 (w), 1527 (m), 1494 (m), 1407 (m), 1468 (m), 1459 (m, sh), 1380 (m), 1287 (m), 1240 (m), 1110 (s), 1077 (m), 1046 (s), 1030 (s, sh), 996 (m) cm-1.
HR-ESI C34H6401ON4P (M+H)+ calcd 719.4355; found 719.4357 -
- The mixture of (3S,4S)-3,4-dihydroxypyrrolidine (0.26 g, 2.56 mmol) and vinylphosphonate from example 7 (0.84 g, 1.28 mmol) in n-BuOH (30 ml) is stirred at 105 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of solvent mixture H1 in ethyl acetate. Obtained compound is dissolved in 0.5 M methanolic HCl (40 ml) and the mixture is stirred at rt for 4 h. The final product is obtained by chromatography on silica gel using linear gradient of H1 in ethyl acetate in 36% overall yield (344 mg, 0.46 mmol) and is lyophilized from water.
A mixture of diastereoisomers ∼ 6:5
1H NMR (499.8 MHz, CD3OD): 0.90 (m, 6H, CH 3(CH2)15); 1.25-1.39 (m, 52H, CH3(CH 2)13CH2CH2-O); 1.55 (m, 4H, CH3(CH2)13CH2 CH2O); 1.95 (m, 4H, OCH2CH2 CH2OC16H33); 2.37-2.52 (m, 4H, CH2P); 3.16 (bm, 2H, H-2b,5b-pyrr); 3.42 (t, 2H, J vic = 6.7, CH3(CH2)13CH2CH2 O); 3.425 (bm, 2H, H-2b,5b-pyrr); 3.43 (t, 2H, J vic = 6.7, CH3(CH2)13CH2CH2 O); 3.515 (m, 4H, CH2N); 3.52, 3.53 (2 × t, 2 × 2H, J vic = 6.1, OCH2CH2CH2 OC16H33); 3.59, 3.92 (2 × bm, 2 × 2H, H-2a,5a-pyrr); 4.14 (m, 2H, H-4'); 4.16 (m, 2H, H-3'); 4.18-4.27 (m, 10H, H-2', OCH2 CH2CH2OC16H33, H-3,4-pyrr); 4.31 (ddd, 1H, J gem = 11.5, J H,P = 7.6, J 5'b,4' = 5.4, H-5'b); 4.36 (m, 2H, H-5'); 4.40 (ddd, 1H, J gem = 11.5, J H,P = 7.3, J 5'a,4' = 2.9, H-5'a); 5.742, 5.746 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.78, 5.80 (2 × d, 2 × 1H, J 1',2' = 3.9, H-1'); 7.68, 7.70 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (125.7 MHz, CD3OD): 14.45 (CH3(CH2)15); 23.32 (d, J C,P = 142.0, CH2P); 23.74 (CH3(CH2)14CH2O); 27.29, 30.48, 30.64, 30.76, 30.79 (CH3(CH2)14CH2O); 31.70, 31.72 (d, J C,P = 6.1, OCH2CH2CH2OC16H33); 33.07 (CH3(CH2)14CH2O); 52.18, 52.22 (CH2N); 60.67, 60.75, 60.98, 61.01 (CH2-2,5-pyrr); 65.33, 65.58 (d, J C,P = 6.7, OCH2CH2CH2OC16H33); 67.16 (d, J C,P = 6.5, CH2-5'); 67.44 (OCH2CH2 CH2OC16H33); 67.45 (d, J C,P = 4.3, CH2-5'); 67.47 (OCH2CH2 CH2OC16H33); 70.77 (CH-3'); 72.16 (CH3(CH2)14 CH2O); 74.55, 74.60 (CH-2'); 75.67, 75.68, 76.04 (CH-3,4-pyrr); 83.29 (d, J C,P = 6.2, CH-4'); 83.40 (d, J C,P = 5.8, CH-4'); 92.83, 92.85 (CH-1'); 103.03, 103.10 (CH-5); 143.12, 143.19 (CH-6); 152.13, 152.14 (C-2); 166.00 (C-4).
31P NMR (202.3 MHz, CD3OD): 26.79, 27.26.
IR vmax(KBr) 3405 (s, vbr), 2694 (w, vbr), 2596 (w, vbr), 2925 (vs), 2854 (s), 1714 (s, sh), 1694 (vs), 1630 (m, sh), 1466 (m), 1458 (m, sh), 1418 (w), 1415 (w), 1385 (m), 1269 (m), 1229 (m, br), 1107 (s), 1053 (m, sh), 1030 (s, br), 1002 (s), 819 (w), 765 (vw), 721 (vw) cm-1.
HR-ESI C34H63O11N3P (M+H)+ calcd 720.4195; found 720.4194 -
- The mixture of (3R,4S,5S)-3,4,5-trihydroxypiperidine (0.04 g, 0.33 mmol) and vinylphosphonate from the example 7 (0.1 g, 0.16 mmol) in n-BuOH (5 ml) is stirred at 105 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. This compound is dissolved in 0.5 M methanolic HCl (20 ml) and the mixture is stirred at rt for 4 h. The final product is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate in 68% overall yield (80 mg, 0.11 mmol) and is lyophilized from water.
A mixture of diastereoisomers ∼ 7:3
1H NMR (499.8 MHz, CD3OD): 0.90 (m, 6H, CH3(CH 2)15); 1.25-1.38 (m, 52H, CH3(CH 2)13CH2CH2-O); 1.55 (m, 4H, CH3(CH2)13CH2 CH2O); 1.93 (m, 4H, OCH2CH2 CH2OC16H33); 2.10-2.18 (m, 4H, CH2P); 2.43, 2.54 (2 × bm, 2 × 4H, H-2,6-pip); 2.75 (m, 4H, CH2N); 3.42, 3.43 (2 × t, 2 × 2H, J vic = 6.6, CH3(CH2)13CH2CH2 O); 3.52, 3.53 (2 × t, 2 × 2H, J vic = 6.1, OCH2CH2CH2 OC16H33); 3.68 (bm, 4H, H-3,5-pip); 3.81 (bm, 2H, H-4-pip); 4.11-4.22 (m, 10H, H-2',3',4', OCH2 CH2CH2OC16H33); 4.23-4.38 (m, 4H, H-5'); 5.746, 5.749 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.84 (d, 2H, J 1',2' = 4.1, H-1'); 7.71, 7.73 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (125.7 MHz, CD3OD): 14.45 (CH3(CH2)15); 23.55, 23.65 (d, J C,P = 138.5, CH2P); 23.75 (CH3(CH2)14CH2O); 27.30, 30.49, 30.63, 30.77, 30.80 (CH3(CH2)14CH2O); 31.79, 31.82 (d, J C,P = 6.2, OCH2CH2CH2OC16H33); 33.08 (CH3(CH2)14CH2O); 51.75 (CH2N); 54.40 (CH2-2,6-pip); 64.72, 64.78 (d, J C,P = 6.5, OCH2CH2CH2OC16H33); 66.37 (d, J C,P = 6.3, CH2-5'); 67.51, 67.55 (OCH2CH2CH2OC16H33); 69.67 (CH-3,5-pip); 70.81, 70.83 (CH-3'); 71.83 (CH-4-pip); 72.17 (CH3(CH2)14CH2O); 74.97, 75.01 (CH-2'); 83.59, 83.63 (d, J C,P = 6.7, CH-4'); 91.84, 91.92 (CH-1'); 103.00 (CH-5); 142.56, 142.60 (CH-6); 152.18, 152.19 (C-2); 166.05 (C-4).
31P NMR (202.3 MHz, CD3OD): 32.91, 33.23.
IR vmax(KBr) 3423 (vs, br), 2924 (s), 2854 (m), 1687 (s), 1632 (m), 1466 (m), 1406 (w), 1380 (w), 1264 (m), 1229 (m), 1116 (m), 1072 (m, sh), 1048 (m), 1029 (m), 996 (m, sh), 766 (vw) cm-1. HR-ESI C35H65O11N3P (M+H)+ calcd 734.4347; found 734.4351 -
- The mixture of (3R,5R)-3,5-dihydroxypiperidine (0.05 g, 0.42 mmol) and vinylphosphonate from the example 7 (0.25 g, 0.38 mmol) in n-BuOH (5 ml) is stirred at 105 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. Obtained compound is dissolved in 0.5 M methanolic HCl (20 ml) and the mixture is stirred at rt for 4 h. The final product is obtained by chromatography on silica gel using linear gradient of H1 in ethyl acetate in 76% overall yield (230 mg, 0.29 mmol) as a white amorphous solid.
A mixture of diastereoisomers ∼ 6:4
1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H, CH3(CH 2)15); 1.25-1.38 (m, 52H, CH3(CH 2)13CH2CH2-O); 1.55 (m, 4H, CH3(CH2)13CH2 CH2O); 1.70 (bm, 4H, H-4-pip); 1.93 (m, 4H, OCH2CH2 CH2OC16H33); 2.12-2.19 (m, 4H, CH2P); 2.36, 2.57 (2 × m, 2 × 4H, H-2,6-pip); 2.66-2.76 (m, 4H, CH2N); 3.42, 3.43 (2 × t, 2 × 2H, J vic = 6.6, CH3(CH2)13CH2CH2 O); 3.51, 3.53 (2 × t, 2 × 2H, J vic = 6.1, OCH2CH2CH2 OC16H33); 4.01 (m, 4H, H-3,5-pip); 4.12-4.24 (m, 10H, H-2',3',4', OCH 2CH2CH2OC16H33); 4.24-4.40 (m, 4H, H-5'); 5.751, 5.754 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.854, 5.856 (2 × d, 2 × 1H, J 1',2' =4.1, H-1'); 7.71, 7.74 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (150.9 MHz, CD3OD): 14.49 (CH3(CH2)15); 23.49, 23.53 (d, J C,P = 138.4, CH2P); 23.75 (CH3(CH2)14CH2O); 27.29, 27.30, 30.49, 30.65, 30.78, 30.81 (CH3(CH2)14CH2O); 31.76, 31.83 (d, J C,P = 6.3, OCH2CH2CH2OC16H33); 33.08 (CH3(CH2)14CH2O); 40.63, 40.65 (CH2-4-pip); 52.02, 52.04 (d, J C,P = 2.0, CH2N); 60.07, 60.10 (CH2-2,6-pip); 64.63, 64.75 (d, J C,P = 6.6, OCH2CH2CH2OC16H33); 65.58 (CH-3,5-pip); 66.27, 66.52 (d, J C,P = 6.2, CH2-5'); 67.51, 67.54 (OCH2CH2CH2OC16H33); 70.77, 70.84 (CH-3'); 72.13, 72.15 (CH3(CH2)14CH2O); 74.99, 75.05 (CH-2'); 83.59, 83.60 (d, J C,P = 6.6, CH-4'); 91.65, 91.66 (CH-1'); 102.99 (CH-5); 142.45, 142.51 (CH-6); 152.16, 152.18 (C-2); 165.99 (C-4).
31P NMR (202.3 MHz, CD3OD): 33.22, 33.53.
IR v max(KBr) 3431 (vs, br), 2924 (m), 2853 (w), 1710 (m, sh), 1693 (m), 1631 (m), 1468 (w), 1414 (vw), 1380 (w), 1265 (w, br), 1230 (w, br, sh), 1113 (m), 1060 (m, br), 1032 (m), 1006 (m), 764 (vw) cm-1.
HR-ESI C35H65O10N3P (M+H)+ calcd 718.4402; found 718.4402 -
- The mixture of (3R,4R)-3,4-dihydroxypyrrolidine (0.26 g, 2.52 mmol) and vinylphosphonate from the example 6 (1.1 g, 1.68 mmol) in n-BuOH (17 ml) is stirred at 100 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. This compound is dissolved in 0.5 M methanolic HCl (50 ml), and the mixture is stirred at rt for 4 h. The final product is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate in 70% overall yield (840 mg, 1.17 mmol) and lyophilized from water.
A mixture of two diastereoisomers ∼ 6:5
1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H, CH 3(CH2)19); 1.25-1.43 (m, 68H, CH3(CH 2)17CH2CH2-O); 1.69 (m, 4H, CH3(CH2)17CH 2CH2O); 2.06-2.23 (m, 4H, CH2P); 2.68 (dd, 4H, J gem = 10.6, J vic = 2.9, H-2b,5b-pyrr); 2.84-2.97 (m, 4H, CH2N); 3.10 (dd, 4H, J gem = 10.6, J vic = 5.0, H-2a,5a-pyrr); 4.07 (m, 4H, H-3,4-pyrr); 4.08-4.13 (m, 4H, CH3(CH2)17CH2CH 2O); 4.13-4.16 (m, 4H, H-3',4'); 4.207, 4.210 (2 × dd, 2 × 1H, J 2',3' = 5.0, J 2',1' = 4.2, H-2'); 4.25 (ddd, 1H, J gem = 11.6, J H,P = 6.8, J 5'b,4' = 4.4, H-5'b); 4.31 (m, 2H, H-5'); 4.35 (ddd, 1H, J gem = 11.6, J H,P = 6.6, J 5'a,4' = 2.7, H-5'a); 5.735, 5.743 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.84, 5.85 (2 × d, 2 × 1H, J 1',2' = 4.2, H-1'); 7.71, 7.74 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (150.9 MHz, CD3OD): 14.46 (CH3(CH2)19); 23.75 (CH3(CH2)17CH2CH2O); 24.85, 24.91 (d, J C,P = 139.8, CH2P); 26.64, 30.28, 30.29, 30.49, 30.69, 30.73, 30.74, 30.77, 30.79, 30.80 (CH3(CH2)17CH2CH2O); 31.55, 31.57 (d, J C,P = 6.0, CH3(CH2)17CH2CH2O); 33.09 (CH3(CH2)17CH2CH2O); 50.81, 50.86 (CH2N); 60.98, 61.02 (CH2-2,5-pyrr); 66.52, 66.54 (d, J C,P = 6.1, CH2-5'); 67.69, 67.73 (d, J C,P = 7.1, CH3(CH2)17CH2CH2O); 70.80, 70.87 (CH-3'); 74.90, 74.95 (CH-2'); 78.19, 78.26 (CH-3,4-pyrr); 83.60 (d, J C,P = 6.5, CH-4'); 91.75, 91.93 (CH-1'); 102.97, 102.99 (CH-5); 142.65 (CH-6); 152.17, 152.20 (C-2); 166.03 (C-4).
31P NMR (202.3 MHz, CD3OD): 31.29, 31.57.
IR v max(KBr) 3354 (m, vbr), 3063 (m), 2927 (vs), 2855 (s), 1694 (vs), 1630 (w, sh), 1467 (m), 1457 (m, sh), 1388 (m), 1267 (m), 1240 (m, sh), 1108 (m), 1075 (m), 1037 (s, br), 999 (s) cm-1.
HR-ESI C35H65O10N3P (M+H)+ calcd 718.44021, found 718.44030. -
- The mixture of (3R,4R)-3,4-dihydroxypyrrolidine (0.3 g, 2.94 mmol) and vinylphosphonate from the example 5 (1.23 g, 1.96 mmol) in n-BuOH (20 ml) is stirred at 100 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. Obtained compound is dissolved in 0.5 M methanolic HCl (50 ml), and the mixture is stirred at rt for 4 h. The final product is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate in 68% overall yield (920 mg, 1.33 mmol) and lyophilized from water.
A mixture of two diastereoisomers ∼ 6:5
1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H, CH3(CH 2)17); 1.25-1.43 (m, 60H, CH3(CH 2)15CH2CH2-O); 1.68 (m, 4H, CH3(CH2)15CH 2CH2O); 2.06-2.19 (m, 4H, CH2P); 2.55 (m, 4H, H-2b,5b-pyrr); 2.73-2.86 (m, 4H, CH2N); 2.99 (m, 4H, H-2a,5a-pyrr); 4.04 (m, 4H, H-3,4-pyrr); 4.05-4.13 (m, 4H, CH3(CH2)15CH2CH 2O); 4.13-4.16 (m, 4H, H-3',4'); 4.199, 4.201 (2 × dd, 2 × 1H, J 2',3'= 4.8, J 2',1' = 4.2, H-2'); 4.24 (ddd, 1H, J gem = 11.6, J H,P = 6.7, J 5'b,4' = 4.2, H-5'b); 4.28 (ddd, 1H, J gem = 11.6, J H,P = 7.4, J 5'b,4'= 2.5, H-5'b); 4.30 (ddd, 1H, J gem = 11.6, J H,P = 5.2, J 5'a,4' = 2.7, H-5'a); 4.34 (ddd, 1H, J gem = 11.6, J H,P = 6.5, J5'a,4' = 2.7, H-5'a); 5.735, 5.745 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.85, 5.86 (2 × d, 2 × 1H, J 1',2' = 4.2, H-1'); 7.71, 7.74 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (150.9 MHz, CD3OD): 14.47 (CH3(CH2)17); 23.75 (CH3(CH2)15CH2CH2O); 25.13, 25.17 (d, J C,P = 139.3, CH2P); 26.65, 30.28, 30.29, 30.49, 30.68, 30.69, 30.72, 30.73, 30.78, 30.79, 30.80 (CH3(CH2)15CH2CH2O); 31.55, 31.57 (d, J C,P = 6.0, CH3(CH2)15CH2CH2O); 33.09 (CH3(CH2)15CH2CH2O); 50.55, 50.57 (d, J C,P = 1.1, CH2N); 61.01, 61.04 (CH2-2,5-pyrr); 66.34, 66.42 (d, J C,P = 6.3, CH2-5'); 66.60, 67.62 (d, J C,P = 6.8, CH3(CH2)15CH2CH2O); 70.80, 70.88 (CH-3'); 74.96, 75.02 (CH-2'); 78.63, 78.68 (CH-3,4-pyrr); 83.63, 83.65 (d, J C,P = 6.7, CH-4'); 91.56, 91.74 (CH-1'); 102.95, 102.98 (CH-5); 142.55, 142.57 (CH-6); 152.18, 152.22 (C-2); 166.03 (C-4).
31P NMR (202.3 MHz, CD3OD): 32.12, 32.43.
IR v max(CHCl3) 3374 (m, vbr), 3063 (m), 2927 (vs), 2855 (s), 1694 (vs), 1630 (m, sh), 1467 (s), 1457 (s, sh), 1388 (m), 1267 (s), 1239 (m, sh), 1109 (s), 1074 (s), 1040 (s, br), 997 (s) cm-1.
HR-ESI C33H61O10N3P (M+H)+ calcd 690.40891, found 690.40891. -
- The mixture of (3R,4R)-3,4-dihydroxypyrrolidine (0.36 g, 3.45 mmol) and vinylphosphonate from the example 4 (1.38 g, 2.3 mmol) in n-BuOH (20 ml) is stirred at 100 °C overnight. The solvent is removed and isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. Obtained compound is dissolved in 0.5 M methanolic HCl (50 ml), and the mixture is stirred at rt for 4 h. The final product is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate in 38% overall yield (566 mg, 0.86 mmol) and is lyophilized from water.
A mixture of two diastereoisomers ∼ 1:1
1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H, CH 3(CH2)15); 1.24-1.43 (m, 52H, CH3(CH 2)13CH2CH2-O); 1.68 (m, 4H, CH3(CH2)13CH 2CH2O); 2.06-2.18 (m, 4H, CH2P); 2.55 (m, 4H, H-2b,5b-pyrr); 2.73-2.86 (m, 4H, CH2N); 2.99 (m, 4H, H-2a,5a-pyrr); 4.04 (m, 4H, H-3,4-pyrr); 4.05-4.12 (m, 4H, CH3(CH2)13CH2CH 2O); 4.12-4.16 (m, 4H, H-3',4'); 4.198, 4.200 (2 × dd, 2 × 1H, J 2',3' = 5.1, J 2',1' = 4.1, H-2'); 4.24 (ddd, 1H, J gem = 11.6, J H,P = 6.5, J 5'b,4' = 4.2, H-5'b); 4.28 (ddd, 1H, J gem = 11.6, J H,P = 7.2, J 5'b,4' = 2.4, H-5'b); 4.30 (ddd, 1H, J gem = 11.6, J H,P = 5.2, J 5'a,4' = 2.7, H-5'a); 4.34 (ddd, 1H, J gem = 11.6, J H,P = 6.6, J 5'a,4' = 2.8, H-5'a); 5.73, 5.74 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.85, 5.86 (2 × d, 2 × 1H, J 1',2' = 4.1, H-1'); 7.71, 7.74 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (150.9 MHz, CD3OD): 14.46 (CH3(CH2)15); 23.75 (CH3(CH2)13CH2CH2O); 25.14, 25.19 (d, J C,P = 139.3, CH2P); 26.65, 30.27, 30.28, 30.50, 30.68, 30.69, 30.72, 30.73, 30.78, 30.81 (CH3(CH2)13CH2CH2O); 31.55, 31.57 (d, J C,P = 6.0, CH3(CH2)13CH2CH2O); 33.09 (CH3(CH2)13CH2CH2O); 50.55, 50.59 (d, J C,P = 1.0, CH2N); 61.02, 61.05 (CH2-2,5-pyrr); 66.34, 66.42 (d, J C,P = 6.3, CH2-5'); 66.60, 67.63 (d, J C,P = 6.8, CH3(CH2)13CH2CH2O); 70.81, 70.89 (CH-3'); 74.96, 75.02 (CH-2'); 78.65, 78.69 (CH-3,4-pyrr); 83.64, 83.66 (d, J C,P = 6.6, CH-4'); 91.58, 91.76 (CH-1'); 102.94, 102.98 (CH-5); 142.57, 142.58 (CH-6); 152.18, 152.22 (C-2); 166.04 (C-4).
31P NMR (202.3 MHz, CD3OD): 32.33, 32.63.
IR v max(CHCl3) 3376 (m, br), 3062 (w), 2927 (vs), 2855 (s), 1693 (vs), 1632 (w, sh), 1466 (m), 1459 (m, sh), 1388 (m), 1267 (s), 1237 (m, sh), 1109 (s), 1074 (s), 1039 (s, br), 997 (s) cm-1.
HR-ESI C31H57O10N3P (M+H)+ calcd 662.37761, found 662.37759. -
- The mixture of (3R,4R)-3,4-dihydroxypyrrolidine (0.42 g, 4.10 mmol) and vinylphosphonate from the example 2 (1.17 g, 2.05 mmol) in n-BuOH (20 ml) is stirred at 100 °C overnight. The solvent is removed and the isopropylidene intermediate is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate. This compound is dissolved in 0.5 M methanolic HCl (50 ml), and the mixture is stirred at rt for 4 h. The final product is obtained by flash chromatography on silica gel using linear gradient of H1 in ethyl acetate in 66% overall yield (862 mg, 1.36 mmol) and is lyophilized from water.
A mixture of two diastereoisomers ∼ 6:5
1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H, CH 3(CH2)13); 1.25-1.43 (m, 44H, CH3(CH 2)11CH2CH2-O); 1.68 (m, 4H, CH3(CH2)11CH 2CH2O); 2.06-2.19 (m, 4H, CH2P); 2.56 (m, 4H, H-2b,5b-pyrr); 2.74-2.88 (m, 4H, CH2N); 3.00 (m, 4H, H-2a,5a-pyrr); 4.04 (m, 4H, H-3,4-pyrr); 4.06-4.13 (m, 4H, CH3(CH2)11CH2CH 2O); 4.13-4.16 (m, 4H, H-3',4'); 4.200, 4.202 (2 × dd, 2 × 1H, J 2',3' = 5.0, J 2',1' = 4.0, H-2'); 4.24 (ddd, 1H, J gem = 11.6, J H,P = 6.7, J 5'b,4' = 4.3, H-5'b); 4.29 (ddd, 1H, J gem = 11.6, J H,P = 7.2, J 5'b,4' = 2.4, H-5'b); 4.31 (ddd, 1H, J gem = 11.6, J H,P = 5.0, J 5'a,4' = 2.6, H-5'a); 4.35 (ddd, 1H, J gem = 11.6, J H,P = 6.6, J 5'a,4' = 2.8, H-5'a); 5.736, 5.745 (2 × d, 2 × 1H, J 5,6 = 8.1, H-5); 5.85, 5.86 (2 × d, 2 × 1H, J 1',2' = 4.0, H-1'); 7.71, 7.74 (2 × d, 2 × 1H, J 6,5 = 8.1, H-6).
13C NMR (150.9 MHz, CD3OD): 14.46 (CH3(CH2)13); 23.75 (CH3(CHZ)11CH2CH2O); 25.11 (d, J C,P = 139.5, CH2P); 25.16 (d, J C,P = 139.0, CH2P); 26.65, 30.27, 30.28, 30.50, 30.68, 30.71, 30.72, 30.78, 30.80, 30.82 (CH3(CH2)11CH2CH2O); 31.55, 31.57 (d, J C,P = 6.0, CH3(CH2)11CH2CH2O); 33.09 (CH3(CH2)11CH2CH2O); 50.57, 50.60 (d, J C,P = 1.0, CH2N); 61.01, 61.03 (CH2-2,5-pyrr); 66.36, 66.42 (d, J C,P = 6.3, CH2-5'); 66.60, 67.63 (d, J C,P = 6.8, CH3(CH2)11CH2CH2O); 70.81, 70.88 (CH-3'); 74.95, 75.01 (CH-2'); 78.60, 78.65 (CH-3,4-pyrr); 83.63, 83.64 (d, J C,P = 6.6, CH-4'); 91.57, 91.75 (CH-1'); 102.95, 102.98 (CH-5); 142.56, 142.57 (CH-6); 152.18, 152.22 (C-2); 166.03 (C-4). 31P NMR (202.3 MHz, CD3OD): 32.08, 32.38.
IR v max(CHCl3) 3374 (m, vbr), 3063 (w), 2927 (vs), 2855 (s), 1694 (vs), 1630 (w, sh), 1467 (m), 1459 (m, sh), 1388 (m), 1267 (m), 1238 (m, sh), 1109 (m), 1074 (m), 1040 (s, br), 997 (s) cm-1.
HR-ESI C29H53O10N3P (M+H)+ calcd 634.34631, found 634.34635. - Antimicrobial activity is assessed using the standard microdilution method determining the minimum inhibitory concentration (MIC) of the tested samples leading to inhibition of bacterial growth. Disposable microtitration plates are used for the tests. The samples were diluted in brain heart infusion broth (Himedia, Laboraties Pvt. Ltd., eská republika) and Mueller-Hinton broth (HiMedia Laboraties, see above) to yield a concentration range between 200 µg/ml and 1.5625 µg/ml. The plates are inoculated with a standard amount of the tested microbe - the inoculum density in each well is equal to 105-6 CFU/ml. The MIC is read after 24/48 hours of incubation at 37°C as the minimum inhibitory concentration of the tested substance that inhibits the growth of the bacterial strains. The minimum bactericidal concentration (MBC) is characterized as the minimum concentration of the sample required to achieve irreversible inhibition, i.e. killing the bacterium after a defined period of incubation. The MBC is examined by the inoculation method. With an applicator, 10 µL are transferred from microplate wells with defined concentrations of the tested sample and inoculated onto the surface of blood agar (Trios, Czech Republic). The MBC is determined as the lowest concentration that inhibited the visible growth of the used bacterium.
- Standard reference bacterial strains (E. faecalis CCM 4224, S. aureus CCM 4223) from the Czech Collection of Microorganisms (CCM), Faculty of Science, Masaryk University Brno, S. agalactiae, B. subtilis, methicilin-resistant S. aureus 4591, fluoroquinolone-resistant S. haemolyticus 16568, vancomycin-resistant E. faecium VanA419/ana and methicilin-resistant S. epidermidis 8700/B strains obtained from the Teaching Hospital Olomouc were used. All tested microorganisms were stored in cryotubes (ITEST plus, Czech Republic) at -80°C.
Table 1 Minimum inhibitory concentration of selected novel lipophosphonoxines against a panel of reference bacterial strains MIC µg/ml Compound prepared in example E. faecalis CCM 4224 S.aureus CCM 4223 B. subtilis S.agalactiae 9 12,5 - 6,25 3,125 10 6,25 25 3,125 3,125 11 12,5 - 6,25 6,25 12 - - - 12,5 13 - - - 6,25 14 - - - 6,25 15 6,25 25-12,5 12,5-6,25 3,125 16 6.25 12,5-6,25 3,125 1,5625 17 12,5 25-12.5 6,25 6,25 Table 2 Minimum inhibitory concentration of selected novel lipophosphonoxines against a panel of resistant bacterial strains MIC µg/ml Compound prepared in example S. aureus MRSA 4591 S. haemolyticus 16568 E. faecium VanA 419/ana S. epidermidis 8700/B 9 - - 6,25 3,125 10 - - 6,25 6,25 11 - - 6,25 6,25 15 - - 6,25 6,25 16 12,5 25-12,5 3,125 3,125 17 25 12,5 12,5 12,5 - Multiresistant bacterial strains isolated from clinical isolates of patients from Teaching Hospital Olomouc: MRSA - (methicilin-resistant S. aureus 4591); S. haemolyticus (fluoroquinolone-resistant strain 16568); E. faecium (vancomycin-resistant strain VanA419/ana); S. epidermidis (methicilin-resistant strain 8700/B).
- In all cases the minimum inhibitory concentration (MIC, the concentration of tested compound at which the growth of 100 % of the tested bacteria was inhibited) is equal to minimum bactericidal concentration (MBC, the concentration of tested compound at which 100% of the tested bacteria is killed). The value of MBC is determined by revaccination of bacteria tested for MIC into an inhibitor free medium and monitoring the potential growth.
- Human immature erythroid progenitors isolated from umbilical cord blood are cultivated in the StemSpan serum-free medium (StemCell Technologies) in the presence of the stem cell factor (SCF, 100 ng/ml), erythropoetin (EPO, 33.34 nkat/ml) and dexamethasone (1 µmol.l-1) (Panzenbock et al. 1998). Cells are plated at 25000 cells/well/20 µl in 384-well plates immediately before addition of lipophosphonoxine compound from particular examples. After 48 hours of incubation at 37 °C in 5% CO2 viability is determined by using the commercial CellTiter-Blue® Cell Viability Assay (Promega, Cat.# G8082). The assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin). Non-viable cells rapidly lose metabolic capacity and thus do not generate a fluorescent signal. The homogeneous assay procedure involves adding the single reagent directly to cells cultured in serum-supplemented medium. After an incubation step, data are recorded.
- Cytotoxicity is determined after 4 hours at 37 °C in 5% CO2 using commercial CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, Cat.# G7892), a fluorometric method for estimating the number of non-viable cells present in multiwell plates. The CytoTox-ONE™ Assay measures the release of lactate dehydrogenase (LDH) from the cells with a damaged membrane. LDH released into the culture medium is measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. The amount of fluorescence produced is proportional to the number of lysed cells.
- The fluorescence intensity is measured using the EnVision plate reader (PerkinElmer). Data are analyzed using GraphPad Prism 5.0 statistical software.
Table 3 Maximum safety concentration of selected novel lipophosphonoxins based on viability of normal erythroid progenitor cells using commercial test CellTiter Blue (Promega Corp., Madison, USA): Compound prepared in example [Max. safety concentration] µg/ml IC50 µg/ml 9 62 93 10 31 46 11 31 40 12 62 141 13 15 45 14 15 29 15 31 38 16 31 44 17 31 47 Table 4 Cytotoxicity of selected newly prepared lipophosphonoxins: Compound prepard in example [Max. safety concentration] µg/ml EC50 µg/ml 9 125 436 14 125 185 15 62 170 16 62 237 17 125 302 - Cytotoxicity of tested compounds against normal human erythroid progenitor cells was determined using commercial test CytoToxOne (Promega Corp., Madison, USA)
- Lipophosphonoxins according to this invention can be used as antibacterial agents in pharmaceutical preparations destined for the treatment of bacterial infections, components of disinfectants and/or selective growth media.
their pharmaceutically acceptable salts and hydrates.
Claims (11)
- Lipophosphonoxins of general formula I,
R2 is selected from a group comprising uracil, thymin, cytosin,
R3 is selected from the group comprising compounds of general formula II and III:
diastereomers and mixtures of diaslereomers thereof, and
their pharmaceutically acceptable salts and hydrates. - A method of preparation of lipophosphonoxins of general formula I according to claim 1 or diastereomers and mixtures od diastereomers thereof, characterized in that it contains steps A-D, in whichA) dimethyl vinylphosphonate is heated overnight with 40% to 70% (v/v) aqueous pyridine at a temperature from 50 to 80 °C affording methyl vinylphosphonate IXB) the obtained methyl vinylphosphonate is esterified with nucleoside X, protected in positions 2'and 3' with a protecting group selected from 2'.3'-isopropylidene, 2',3'-bis(diniethoxytrityl), 2',3'-dibenzoyl, or 2',3'-diacetyl, and possessing free 5'-hydroxyl groupC) the vinylphosphonic acid XI is esterified by alcohol using triisopropylbenzenesulfonylchloride as condensing agent and under catalysis by N-methylimidazole in a solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran, dichloroethane, chloroform and dichloromethane, and the thereby obtained vinylphosphonate XIID) Michael addition of secondary amine to the vinyl function of the vinylphosphonate XII is performed by heating in an organic solvent selected from the group comprising acetonitrile, dioxane, tetrahydrofuran, dichloroechane, chloroforme, etanol, propanol, isopropanol a n-butanol at temperature from 80 to 120 °C for 4 to 12 hours, followed by removal of the protecting groups on nucleoside; the product is subsequently isolated by chromatography on silica gel using linear gradient of mixture of ethyl acetate:acetone:ethanol:water in volume ratios 4:1:1:1, in ethyl acetate; or it is re-purified by reverse phase high performance liquid chromatography.
- The method of preparation according to claim 2, characterized in that heating overnight with aqueous pyridine in step A) and/or B) is carried out at 60 °C and the concentration of the pyridine solution is 60% (v/v).
- The method of preparation according to claim 2, characterized in that Michael addition in the step D is carried out in n-butanol at 105 °C. preferably overnight.
- The method of preparation according to claim 2, characterized in that esterification in the steps B and/or C is preferably carried out in dichloromethane.
- The method of preparation according to claim 2, characterized in that in the step D), the nucleoside protecting groups selected from 2',3'-bis(dimethoxytrityl), 2',3'-dibenzoyl and 2',3'-diacetyl are removed by ethanolic methylamine or aqueous ammonia.
- The method of preparation according to claim 2, characterized in that in the step D), the 2',3'-isopropylidene protecting group is removed by treatment with 0,5 mol.1-1 hydrochloride in methanol at room temperature overnight.
- The lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds according to claim 1 for use as medicaments.
- Lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds according to claim 1 for use as antibacterial medicaments.
- Lipophoshonoxins of general formula I or their diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds according to claim 1 for use as an active component of disinfectants and/or selective growth media for in vitro cultivations.
- Antibacterial medicament, disinfectant and/or selective growth medium containing as an active ingredient at least one lipophoshonoxin of general formula I or their diastereomers and mixtures of such diastereomers or pharmaceutically acceptable salts and hydrates thereof and/or mixtures of such compounds according to claim 1.
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